As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, Liver Receptor Homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases. Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly-discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of inflammatory bowel disease, the new agonist increases expression of LRH-1-conrolled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility. glucose metabolism suggests therapeutic potential for LRH-1 agonists in metabolic diseases such as nonalcoholic fatty liver disease, type II diabetes, and cardiovascular disease. Indeed, the phospholipid LRH-1 agonist dilauroylphosphatidylcholine (DLPC; PC 12:0/12:0) improves glucose tolerance, insulin sensitivity, and triglyceride levels in obese mice 6 . These anti-diabetic effects occur in an LRH-1-dependent manner and have been primarily attributed to a reduction of de novo lipogenesis 6 . In addition, targeting LRH-1 in the gut has therapeutic potential for inflammatory bowel disease, where LRH-1 overexpression ameliorates disease-associated inflammation and cell death 10 .While small molecule LRH-1 modulators are highly sought, the large and lipophilic LRH-1 binding pocket has been extremely challenging to target. A promising class of agonists developed by Whitby and colleagues features a bicyclic hexahydropentalene core scaffold [11][12] . The beststudied of this class, named RJW100, was discovered as a part of an extensive synthetic effort to improve acid stability and efficacy of a related compound, GSK8470 12 (Figure 1A). We recently determined the crystal structure of LRH-1 bound to RJW100 and made a surprising discovery: it exhibits a completely different binding mode than GSK8470, such that the bicyclic cores of the two agonists are perpendicular to each other (Figure 1A) 13 . As a result, the two compounds use different mechanisms to activate LRH-1 but exhibit similar activation profiles in luciferase reporter assays 13 . A tendency for ligands in this class to bind unpredictably in the hydrophobic pocket has likely been a confounding factor in agonist design; however, insights from the LRH-1-RJW100 structure have provided new strategies to improve activity.In the LRH-1-RJW100 crystal structure, the ligand hydroxyl group contacts a network of water molecules deep in the ligand binding pocket (Figure 1B). This water network coordinates a

As a key regulator of metabolism and inflammation, the orphan nuclear hormone receptor, Liver Receptor Homolog-1 (LRH-1), has potential as a therapeutic target for diabetes, nonalcoholic fatty liver disease, and inflammatory bowel diseases. Discovery of LRH-1 modulators has been difficult, in part due to the tendency for synthetic compounds to bind unpredictably within the lipophilic binding pocket. Using a structure-guided approach, we exploited a newly-discovered polar interaction to lock agonists in a consistent orientation. This enabled the discovery of the first low nanomolar LRH-1 agonist, one hundred times more potent than the best previous modulator. We elucidate a novel mechanism of action that relies upon specific polar interactions deep in the LRH-1 binding pocket. In an organoid model of inflammatory bowel disease, the new agonist increases expression of LRH-1-conrolled steroidogenic genes and promotes anti-inflammatory gene expression changes. These studies constitute major progress in developing LRH-1 modulators with potential clinical utility.de novo lipogenesis 6 . In addition, targeting LRH-1 in the gut has therapeutic potential for inflammatory bowel disease, where LRH-1 overexpression ameliorates disease-associated inflammation and cell death 10 .While small molecule LRH-1 modulators are highly sought, the large and lipophilic LRH-1 binding pocket has been extremely challenging to target. A promising class of agonists developed by Whitby and colleagues features a bicyclic hexahydropentalene core scaffold 11-12 . The beststudied of this class, named RJW100, was discovered as a part of an extensive synthetic effort to improve acid stability and efficacy of a related compound, GSK8470 12 ( Figure 1A). We recently determined the crystal structure of LRH-1 bound to RJW100 and made a surprising discovery: it exhibits a completely different binding mode than GSK8470, such that the bicyclic cores of the two agonists are perpendicular to each other ( Figure 1A) 13 . As a result, the two compounds use different mechanisms to activate LRH-1 but exhibit similar activation profiles in luciferase reporter assays 13 . A tendency for ligands in this class to bind unpredictably in the hydrophobic pocket has likely been a confounding factor in agonist design; however, insights from the LRH-1-RJW100 structure have provided new strategies to improve activity.In the LRH-1-RJW100 crystal structure, the ligand hydroxyl group contacts a network of water molecules deep in the ligand binding pocket ( Figure 1B). This water network coordinates a small group of polar residues (e.g. Thr352, His390, and Arg393) in an otherwise predominantly hydrophobic pocket. The endo RJW100 diastereomer adopts a nearly identical pose and makes the same water-mediated contact with Thr352, supporting the idea that this interaction is a primary driver of ligand orientation 13 . Using both a RJW100 analog lacking a hydroxyl group and a LRH-1 Thr352Val mutation, we demonstrated that this interaction is required for RJW100-mediated activation o...

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